The Mycobacteria are a genus of bacteria which are acid-fast, non-motile, grampositive rods. The genus comprises several species which include, but are not limited to, Mycobacterium africanum, M. avium, M. bovis, M. bovis-BCG, M. chelonae, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. microti, M. scrofulaceum, M. paratuberculosis and M. tuberculosis (M.tb). Certain of these organisms are the causative agents of disease. For the first time since 1953, cases of mycobacterial infections are increasing in the United States. Of particular concern is M. tuberculosis, which infects one third of the world's population and is the etiological agent of tuberculosis. Many new cases of mycobacterial infection are related to the AIDS epidemic, which provides an immune compromised population which is particularly susceptible to infection by Mycobacteria. The World Health Organization also estimates that approximately 3 million people will die from tuberculosis annually. Although effective antibiotic treatments are available for tuberculosis, the recent emergence of multiple-drug resistant strains of M. tuberculosis poses a serious public health concern. M. tuberculosis and other mycobacteria which are closely related to it (M. bovis, M. africanum, M. bovis BCG and M. microti) are referred to as the "TB complex." Mycobacterial infections caused by species other than tuberculosis are also increasing as a result of recent increases in the number of immune compromised patients. For example, M. avium, M. kansasii and other non-tuberculosis mycobacteria are found as opportunistic pathogens in patients infected with HIV as well as in in other immune compromised patients. These and other non-TB complex species are referred to as "mycobacteria other than tuberculosis" (MOTT).
The first isolation of a bacteriophage which infected a mycobacterium (mycobacteriophage) was reported in 1947. This mycobacteriphage infected M. tuberculosis. Since that time, a large number of different mycobacteriophage have been isolated and characterized. The host range of mycobacteriophage varies greatly, with some capable of infecting only a single species. Others (e.g., D29) have a very broad range of mycobacterial hosts. The different host ranges of certain mycobacteriophage have been utilized in a phage typing system for M. tuberculosis (Crawford and Bates. 1984. The Mycobacteria--A Sourcebook Vol. 15 G. P. Kubica and L. G. Wagner, eds. Marcel Dekker, Inc., New York). In addition, the isolation and characterization of mycobacteriophage has made possible their use as cloning vectors for introducing genes into mycobacteria, in some cases species-specifically (W. R. Jacobs, et al. 1989 Rev. Inf. Dis. 11 (Supp. 2):S404-S410).
The recent increase in the number of clinical isolates of tuberculosis which are resistant to at least one of the antibiotics normally used to treat the disease (e.g., isoniazid, rifampin or streptomycin) has resulted in a corresponding increase in the number of fatalities in both immunocompetent and immunocompromised individuals. Because M.tb. grows very slowly (doubling time 20-24 hrs.), conventional methods for identifying this organism and determining drug susceptibility require 2-18 weeks. Conventional diagnosis of mycobacterial infections generally relies on acid-fast staining and cultivation of the organism, followed by biochemical and morphological assays to confirm the presence of mycobacteria and identify the species. These procedures are time-consuming, and a typical diagnosis using conventional culture methods can take as long as six weeks. Automated culturing systems such as the BACTEC.TM. system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) can decrease the time for detection of mycobacteria to one to two weeks. Once detected, culturing these slow-growing microorganisms in the presence of antibiotics to determine their drug susceptibility requires several additional weeks. There is still a need to even further reduce the time required for diagnosing mycobacterial infections and determining antibiotic susceptibility in order to allow prompt, informed treatment of M.tb. infections.
The BACTEC TB System provides one means for determining whether or not a positive mycobacterial culture is the result of TB complex mycobacteria or mycobacteria other than tuberculosis (MOTT). This is important information for the initial diagnosis of tuberculosis, and shortens the time required for determining the species present in a positive mycobacterial culture. The BACTEC TB identification scheme relies on a combination of three tests, namely, morphology on smear, growth characteristics and the NAP (p-nitro-.alpha.-acetylamino-.beta.-hydroxy-propiophenone) TB differentiation test. To improve identification of TB complex species, it is highly desirable to shorten the length of time required to perform such distinguishing tests.
Of particular interest in this regard is the recent development of a diagnostic assay employing recombinant mycobacteriophage. The cDNA encoding firefly luciferase (FFluc) has been inserted into the genomes of mycobacteriophage for use as a reporter gene in antibiotic susceptibility testing of mycobacteria, i.e., as an in vivo measure of cell viability after exposure to antibiotics. W. R. Jacobs, et al. (1993) Science 260:819 and WO 93/16172. Luciferase is useful as a biological reporter or signal generating molecule because it catalyzes the reaction of luciferin with adenosine triphosphate (ATP), resulting in the production of light. Inhibition of culture growth results in reduced or absent light production from the cloned luciferase gene. This effect has been attributed to reduced amounts of ATP (required for the luciferase reaction) in antibiotic-sensitive cells, which exhibit reduced metabolic activity in the presence of an anti-mycobacterial antibiotic, but many other metabolic functions may be affected as well.
Certain mycobacteriophage (e.g., TM4 or phAE40) have been characterized as preferentially infecting species of the TB complex. However, none of these phage are perfectly TB complex-specific and are capable of efficiently infecting certain MOTT species as well. As a result, reporter mycobacteriophage constructed in, for example, TM4 also produce high levels of signal in certain MOTT species. This produces false-positives which are unacceptable for clinical detection and identification of TB complex mycobacteria. A reporter mycobacteriophage which is truly specific for TB-complex organisms is therefore highly desirable for development of a useful diagnostic test.
Mycobacteriophage DS6A was originally isolated from stockyard soil by W. B. Redmond and J. C. Cater (1960. Amer. Rev. Resp. Dis. 82:781-786). They found that DS6A was lytic on M. tuberculosis and M. bovis strains but did not lyse any other mycobacterial strain tested. Based on its unique host range, DS6A was included in the Mycobacterial Typing Phage panel for typing and epidemiological analysis of M. tuberculosis isolates. DS6A has subsequently been tested on over 8,000 strains of M. tuberculosis and M. bovis and never failed to form plaques. A few strains of M. africanum have also been tested and found to be infected by DS6A. While DS6A was previously known primarily as an M. tuberculosis and M. bovis-specific phage, its ability to infect other TB complex species and the degree of TB-complex specificity has never been fully determined. The present invention, by virtue of the discovery that DS6A is truly specific for TB complex mycobacteria, for the first time allows specific detection of TB complex species in a reporter mycobacteriophage (RM) assay. No truly TB-complex-specific mycobacteriophage were previously known, so it was unexpected that the full host range testing described herein would reveal this property in DS6A. Use of DS6A as a reporter mycobacteriophage therefore provides a truly TB complex-specific diagnostic test, eliminates the false-positives of prior art RM assays for TB complex mycobacteria and provides more accurate identification of TB complex organisms using a reporter mycobacteriophage.
As used herein, the term "reporter gene" refers to a gene which can be expressed to produce a gene product which directly or through further reaction generates a detectable signal. Infected cells are detected by means of the reporter gene signal associated with the infected cells. This signal can be used to detect or identify cells carrying the reporter gene, either on a plasmid or inserted into the genome of the cell. Many reporter genes are known in the art, as are methods for using their expression to generate a detectable signal. Examples of reporter genes known in the art are the gene encoding firefly luciferase (resulting in a luminescent signal upon reaction with luciferin) and the gene encoding .beta.-galactosidase (resulting in a colored or fluorescent signal upon reaction with appropriate enzyme substrates). A mycobacteriophage carrying a reporter gene is referred to herein as a "reporter mycobacteriophage" or "RM."